PROJECT SUMMARY Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is a growing public health burden as the population ages. Dose-intensive induction and consolidation chemotherapy dramatically reduces tumor burden and induces clinical complete remission in the majority of younger individuals. However, AML patients rarely achieve durable response and typically relapse with chemoresistant disease. Thus, improved therapeutic approaches are imperative. AML is extremely heterogeneous in terms of clinical behavior as well as molecular alterations. The field increasingly utilizes molecular profiling to provide prognostic predictors of outcome and identify targets for selective inhibitors. The overarching hypothesis of this Program Project is that sphingolipid metabolism is dysregulated in AML and represents a promising target for therapy. Acid ceramidase (AC) is a central mediator in sphingolipid metabolism that controls the levels of the pro-apoptotic lipid ceramide and pro-survival lipid sphingosine 1-phosphate (S1P). AC expression and enzymatic activity are significantly elevated in primary AML samples. The premise of the project is supported by our recent data demonstrating that patients exhibiting high AC activity also showed reduced progression-free and overall survival. AC inhibitors and gene knockdown exhibited therapeutic benefit in human AML cell lines, primary AML samples, and murine AML models, thereby validating AC as a promising target in this disease. Two specific aims will be pursued to explore the hypothesis that new AC inhibitors will exhibit increased potency in AML and that molecular alterations modify the susceptibility to AC targeting agents. Specific Aim 1 will characterize the ability of new AC inhibitors to alter sphingolipid metabolism and the mechanism whereby they induce killing in AML cell lines and patient samples. Nano-encapsulation strategies will be optimized to enhance in vivo drug delivery of these compounds. The efficacy of these inhibitors will then be tested in combination with C6-ceramide nanoliposomes (CNL), the Bcl-2 inhibitor venetoclax, and the AraC/venetoclax combinatorial regimen that is investigated across all Projects. We demonstrate that AC inhibitors increase the efficacy of each of these agents. These approaches will be applied to state-of-the-art human AML xenograft models to demonstrate preclinical efficacy and will be compared to standard-of-care chemotherapy models. Specific Aim 2 will determine the relationship between AML molecular subtypes, AC activity, and sensitivity to AC inhibitors. These studies will be completed across diverse AML cell lines and primary patient samples and will include cooperative analysis together with the Systems Metabolomics Core (Core C). Next, the link between AC and mutations (NPM1c, FLT3-ITD, DNMT3AR882H) that are frequently detected in AML will be characterized. These studies will utilize genetically engineered cell lines and mouse models, which also provide the opportunity to validate AC targeting in vivo in the context of defined molecular drivers. The proposed studies will facilitate future clinical application of AC inhibitors in AML by identifying the optimal therapeutic agent as well as susceptible patient populations.